Routine vaccinations used in veterinary practice have had a highly beneficial impact on the health and welfare of livestock and companion animals. Poultry vaccines can be administered via different routes and by various methods. For example, a post-hatch spray vaccination method has been widely used. This method can mass-immunize day old chicks through aerosol spray. Also, live or attenuated vaccines can be administered to poultry through traditional method, i.e., by subcutaneous injection to chicks, rearing stock and breeders. Furthermore, poultry vaccines can be delivered via eye drops and/or intranasal routes during brooding of chicks. Finally, and most prevalently, vaccines can be administered to poultry via drinking water. This vaccination method has the advantage of low cost, but its effectiveness, particularly against some infections, is limited due to less control of vaccination.
Most recently, some poultry vaccines have been administered to eggs through an in ovo injection method. A patented EMBREX INOVOJECT® system has been used to facilitate this kind of injection. The first in-ovo vaccination machine for use on chicken hatching eggs was developed by Embrex, Inc., of Raleigh, N.C. in the late 1980s. (See U.S. Pat. Nos. 5,056,464 and 5,699,751).
This in-ovo machine is currently used in about 80% of the U.S. broiler hatcheries, primarily for administering Marek's disease (MD) vaccines. The popularity of this machine, which has proven to be safe and effective in vaccination of chicks against MD, is also being used increasingly to administer infectious bursal disease (IBD) and Newcastle disease (ND) vaccines.
In ovo vaccination of virus-containing vaccines was extensively described by Sharma et al. (U.S. Pat. No. 4,458,630). In particular, it teaches that live Marek's disease virus can be injected into amniotic fluid within the egg, whereafter the embryo is infected and the vaccine virus replicates to a high titer which induces the formation of protective antibodies in the treated embryo. (See Sharma (1985), Avian Diseases 29, 1155, 1167–68).
It is well-known in the worldwide poultry business that certain viral diseases, such as Marek's disease virus (MDV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian encephalomyelitis (AEV), chick anemia virus (CAV), Fowlpox virus (FPV), avian influenza virus (AIV), reovirus, avian leukosis virus (ALV), reticuloendotheliosis virus (REV), avian adenovirus and hemorrhagic enteritis virus (HEV), may cause major outbreak and result in significant economic losses in the commercial poultry industry. Among them, MDV, IBDV, NDV and IBV, are particularly important due to their virulent nature.
Marek's Disease (MD) is a malignant, lymphoproliferative disorder disease that occurs naturally in chickens. The disease is caused by a herpesvirus: Marek's Disease Virus (MDV). MD is ubiquitous, occurring in poultry-producing countries throughout the world. Chickens raised under intensive production systems will inevitably suffer losses from MD. The symptoms of MD appear widely in the nerves, genital organs, internal organs, eyes and skin of the infected birds, causing motor trouble (due to paralysis when the nerves have been affected), functional trouble of the internal organs (due to tumors), and chronic undernourishment (if the internal organs are attacked by the virus). MD affects chickens from about 6 weeks of age, occurring most frequently between ages of 12 and 24 weeks.
At of this time, there are no methods of treating MD. The control of the disease is based primarily on management methods such as insolating growing chickens from sources of infection, the use of genetically resistant stock, and vaccination. However, management procedures are normally not cost-effective and the progress has been disappointing with respect to the selection of poultry stock with increased genetically controlled resistance. Nowadays, control of MD is almost entirely based on vaccination.
Infectious bursal disease virus (IBDV) is responsible for a highly contagious immunosuppressive disease in young chickens which causes significant losses to the poultry industry worldwide (See Kibenge (1988), J. Gen. Virol., 69:1757–1775). Infection of susceptible chickens with virulent IBDV strains can lead to a highly contagious immunosuppressive condition known as infectious bursal disease (IBD). Damage caused to the lymphoid follicles of the bursa of Fabricius and spleen can exacerbate infections caused by other agents and reduce a chicken's ability to respond to vaccination as well (See Cosgrove (1962), Avian Dis., 6:385–3894).
IBDV is a member of the Birnaviridae family and its genome consists of two segments of double-stranded RNA (See Dobos et al (1979), J. Virol., 32:593–605). The smaller segment B (about 2800 bp) encodes VP1, the dsRNA polymerase. The larger genomic segment A (about 3000 bp) encodes a 110 kDa precursor polypeptide in a single open reading frame (ORF) that is processed into mature VP2, VP3 and VP4 (See Azad et al (1985), Virology, 143:35–44). From a small ORF partly overlapping with the polypeptide ORF, segment A can also encode VP5, a 17-kDa protein of unknown function (See Kibenge et al (1991), J. Gen. Virol. 71:569–577).
While VP2 and VP3 are the major structural proteins of the virion, VP2 is the major host-protective immunogen and causes induction of neutralizing antibodies (See Becht et al. (1988) J. Gen. Virol., 69:631–640; Fahey et al. (1989), J. Gen. Virol., 70:1473–1481). VP3 is considered to be a group-specific antigen because it is recognized by monoclonal antibodies (Mabs) directed against VP3 from strains of both serotype 1 and 2 (See Becht et al (1988), J. Gen. Virol., 69:631–640). VP4 is a virus-coded protease and is involved in the processing of the precursor protein (See Jagadish et al. (1988), J. Virol., 62:1084–1087).
In the past, control of IBDV infection in young chickens has been achieved by live vaccination with avirulent strains, or principally by the transfer of maternal antibody induced by the administration of live and killed IBDV vaccines to breeder hens. Unfortunately, in recent years, virulent variant strains of IBDV have been isolated from vaccinated flocks in the United States (See e.g., Snyder et al. (1988), Avian Dis., 32:535–539; Van der Marel et al. (1990), Dtsch. Tierarztl. Wschr., 97:81–83), which drastically undermine the effectiveness of using live vaccination for IBDV.
Efforts to develop a recombinant vaccine for IBDV have also been made, and the genome of IBDV has been cloned (See Azad et al (1985) “Virology”, 143:35–44). The VP2 gene of IBDV has been cloned and expressed in yeast (See Macreadie et al. (1990), Vaccine, 8:549–552), as well as in recombinant fowlpox virus (See Bayliss et al (1991), Arch. Virol., 120:193–205). When chickens were immunized with the VP2 antigen expressed from yeast, antisera afforded passive protection in chickens against IBDV infection. When used in active immunization studies, the fowlpox virus-vectored VP2 antigen afforded protection against mortality, but not against damage to the bursa of Fabricius.
Newcastle disease virus (NDV) is an enveloped virus containing a linear, single-strand, nonsegmented, negative sense RNA genome. Typically, virus families containing enveloped single-stranded RNA of the negative-sense genome are classified into groups having non-segmented genomes (e.g., Paramyxoviridae and Rhabdoviridae) or those having segmented genomes (e.g., Orthomyxoviridae, Bunyaviridae and Arenaviridae). NDV, together with parainfluenza virus, Sendai virus, simian virus 5, and mumps virus, belongs to the Paramyxoviridae family.
The structural elements of the NDV include the virus envelope which is a lipid bilayer derived from the cell plasma membrane. The glycoprotein, hemagglutinin-neuraminidase (HN) protrude from the envelope allowing the virus to contain both hemagglutinin and neuraminidase activities. The fusion glycoprotein (F), which also interacts with the viral membrane, is first produced as an inactive precursor, then cleaved post-translationally to produce two disulfide linked polypeptides. The active F protein is involved in penetration of NDV into host cells by facilitating fusion of the viral envelope with the host cell plasma membrane. The matrix protein (M), is involved with viral assembly, and interacts with both the viral membrane as well as the nucleocapsid proteins.
The main protein subunit of the NDV nucleocapsid is the nucleocapsid protein (NP) which confers helical symmetry on the capsid. In association with the nucleocapsid are the P and L proteins. The phosphoprotein (P), which is subject to phosphorylation, is thought to play a regulatory role in transcription, and may also be involved in methylation, phosphorylation and polyadenylation. The L gene, which encodes an RNA-dependent RNA polymerase, is required for viral RNA synthesis together with the P protein. The L protein, which takes up nearly half of the coding capacity of the viral genome is the largest of the viral proteins, and plays an important role in both transcription and replication.
The replication of all negative-strand RNA viruses, including NDV, is complicated by the absence of cellular machinery required to replicate RNA. Additionally, the negative-strand genome can not be translated directly into protein, but must first be transcribed into a positive-strand (mRNA) copy. Therefore, upon entry into a host cell, the virus can not synthesize the required RNA-dependent RNA polymerase. The L, P and NP proteins must enter the cell along with the genome on infection. Both the NDV negative strand genomes (vRNAs) and antigenomes (cRNAs) are encapsidated by nucleocapsid proteins; the only unencapsidated RNA species are virus mRNAs. The cytoplasm is the site of NDV viral RNA replication, just as it is the site for transcription. Assembly of the viral components appears to take place at the host cell plasma membrane and mature virus is released by budding.
In U.S. Pat. No. 5,427,791, Ahmad et al. describe the embryonal vaccination against NDV, which requires the modification of the viruses through the use of ethyl methane sulfonate (EMS). However, EMS is a mutagen so that the vaccine prepared by the use of EMS is suspected to act as a mutagen as well, which is undesirable for regular administration of the vaccine. Nevertheless, without the modification with EMS, the NDV vaccine cannot be applied for in ovo vaccination as almost all of the embryos will die upon injection of the eggs with the unmodified virus.
Infectious bronchitis virus (IBV), the prototype of the family Coronaviridae, is the etiological agent of infectious bronchitis (IB). The virus has a single-stranded RNA genome, approximately 20 kb in length, of positive polarity, and is usually about 80–100 nm in size, being round with projecting 20 nm spikes. IBV is the causative agent of an acute, highly contagious disease in chickens of all ages, affecting the respiratory, reproductive and renal systems.
IBV contains three structural proteins: the spike (S) glycoprotein, the membrane glycoprotein, and the nucleocapsid protein. The spike glycoprotein is so called because it is present in the teardrop-shaped surface projections or spikes protruding from the lipid membrane of the virus. The spike protein is believed likely to be responsible for immunogenicity of the virus, partly by analogy with the spike proteins of other corona-viruses and partly by in vitro neutralization experiments (See, e.g., D. Cavanagh et al. (1984), Avian Pathology, 13, 573–583). There are two spike glycoproteins, which are S1 (90,000 daltons) and S2 (84,000 daltons). The polypeptide components of the glycopolypeptides S1 and S2 have been estimated after enzymatic removal of oligosaccharides to have a combined molecular weight of approximately 125,000 daltons. It appears that the spike protein is attached to the viral membrane by the S2 polypeptide.
IBV has been wide-spread in countries where an intensive poultry industry has been developed. Young chickens up to 4 weeks of age are most susceptible to IBV, infection leading to high rates of morbidity and to mortality resulting from secondary bacterial infection. Infection also results in a drop in egg production, or failure to lay at full potential, together with an increase in the number of down-graded eggs with thin, misshapen, rough and soft-shells produced, which can have a serious economic effect.
Administering live vaccines to a developing chick in the egg (in-ovo) has proven to be a fast (40,000 eggs per hour), effective (100% of the eggs receive the vaccine), and labor saving ($100,000 per year per hatchery) method to vaccinate baby chicks against certain diseases before they hatch.
Recently, Embrex, Inc. has developed a live viral vaccine called VNF® (Viral Neutralizing Factor). The VNF contains an antibody (immunoglobulin) specific for the virus used in the vaccine. This specific antibody is mixed in an appropriate ratio with the vaccine virus to form a virus-antibody complex (immune complex) vaccine. The amount of the antibody in a complex vaccine is so small that it does not provide passive immunity or neutrialize the vaccine virus. On the other hand, the amount of antibody added to the vaccine virus is enough to delay by several days the normal course of vaccine virus replication.
This delayed vaccine virus replication allows for the safe in ovo administration of moderately attenuated vaccine viruses in young animals. Moderately attenuated vaccine viruses are better at overcoming maternal immunity and at stimulating strong protective immune responses than highly attenuated vaccine viruses. Therefore, the virus-antibody complex vaccine technology allows for the safe hatchery use of vaccine viruses that are not eliminated by maternal antibody. Vaccination in the hatchery (either in ovo or at hatch) is better controlled and more uniform than field vaccination. Thus, the virus-antibody complex vaccine technology improves vaccine efficacy as well as safety.
In the invention to be presented in the following sections, immuno-stimulatory nucleotides, particularly a DNA sequence, specific for avian viral vaccines (collectively “vaccine accelerator factor” and abbreviated as “VAF”) will be introduced. The VAF can be either co-administered with or separately administered from the commercially available vaccines. The VAF can be either a single or multiple DNA constructs, each containing a single DNA molecule which is a viral gene or a fragment thereof. Additionally, the VAF can be a multivalent DNA construct, which contains two or more viral genes or fragments thereof linking together in one DNA construct. The viral genes or fragments used in preparation of the VAFs are those that encode viral peptides which are antigenic to and can induce both the humoral and the cellular immune system in a host. The VAFs are preferably applied in ovo in an appropriate quantity.
Although the VAFs share the same advantages as the VNF, i.e., to improve vaccine efficacy as well as safety, the VAFs differ from the antibody used in Embrex VNF. Most importantly, the VAF stimulates immunological responses, such as antibody induction, T-cell activation with cytokine secretion, and the production of cytotoxic T lymphocytes, while the antibody in VNF acts to delay the normal course of vaccine virus replication. The VAF allows the immune system to respond to the live vaccines much faster and enables the live vaccines to generate much higher titres of antibody than without the VAF. Also, the VAF's effect on stimulating the immune response is not influenced by maternal immunity. Thus, the VAF, in sum, improves pre-hatch immune response, which in turn improves life long poultry health and reduces vaccination costs.
Commercial poultry are an extremely important source for food. Through the combined use of VAF and appropriate vaccination program, the potential losses caused by infections of commercial flocks can be minimized.